Intervertebral discs, the disc-shaped cartilaginous bodies interposed between adjacent vertebrae, are each composed of an outer annulus fibrosus surrounding the inner nucleus pulposus, and comprise cartilaginous components such as proteoglycans, aggrecans, type II collagen and the like. Intervertebral discs lose their elasticity due to reduction of water content in discs occurring primarily as a result of aging, with concomitant loss of cartilaginous components and build-up of fibrous tissue, which leads to destruction of the double structure of the inner nucleus pulposus and the outer annulus fibrosus. This change is referred to as “degenerative intervertebral disc”. Degenerative intervertebral disc progresses with increasing age beginning in the third decade, with degeneration of virtually all intervertebral disc tissue occurring by age 70. Herniated disc is caused when degenerative nucleus pulposus ruptures the fragile fibrous tissue and protrudes into the spinal canal, compressing against the nerve root and cauda equina and producing pain or paralysis. Lumbago can also be caused by abnormal bone metabolism, external injury, tumor or the like, but usually results from degeneration of intervertebral discs.
Magnetic Resonance Imaging (MRI) is widely used for diagnosis of degenerative intervertebral discs. Because it does not involve exposure as with X-rays, it can be used for frequent examination. Sequential imaging has confirmed a natural resorption mechanism whereby the volume of herniated discs decreases with time. Also, use of the contrast agent gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) has shown abundant neovascularization in herniated discs when an imaging effect is seen in the herniated intervertebral disc tissue, suggesting that more natural resorption is occurring. This principle can be utilized for diagnosis and prognosis.
Immunohistological examination of extracted human surgical samples of herniated disc masses has revealed increased neovascularization and infiltration of numerous inflammatory cells consisting primarily of macrophages, in the cartilage matrix of herniated disc masses (Haro et al., Spine 21 (1996), 1647-1652). It has been confirmed that the infiltrating macrophages and intervertebral disc chondrocytes abundantly express the matrix metalloproteinases (MMPs) MMP-3 and MMP-7 (Haro et al., Spine 22 (1997), 1098-1104 and Haro et al. J. Spinal. Disorders 12 (1999), 245-249). MMPs are enzymes which function mostly in the neutral range and are physiologically expressed intraarticularly and in the intervertebral disc tissue. Proteoglycans and aggrecans, major cartilage tissue components, are the substrates of MMP-3 and MMP-7. It is therefore assumed that MMP-3 and MMP-7 play important roles in the degradation and resorption of herniated discs.
When wild-type mouse intervertebral discs are co-cultured with active macrophages, reduction in intervertebral disc mass is greater than with co-culturing of macrophages and intervertebral discs derived from MMP-3 deficient mouse. Experiments using wild-type mice and MMP-3 deficient mice have confirmed that MMP-3 has a chemotactic effect on macrophages causing them to migrate toward the intervertebral discs (MMP-3 functions as a chemotactic factor for macrophages) (Haro et al., J. Clin. Invest. 105(2) (2000), 133-141).
MMP-7 elicits the inflammatory cytokine TNF-α, and TNF-α has been reported to promote production of MMP-3 in intervertebral disc cells (Haro et al., J. Clin. Invest. 105(2) (2000), 143-150). However, it has never been attempted to use human-derived proteases such as MMP-3 and MMP-7 expressed in human herniated discs as therapeutic agents.
Surgical treatment for herniated discs ordinarily attempts to achieve nerve decompression by removal of the herniated disc mass. However, because the condition is common among adolescent and middle age groups and relatively frequent among sport athletes, a non-invasive treatment has been sought in order to avoid surgery.
Treatment by injection of enzymes such as plant-extracted chymopapain into herniated discs is already practiced in the U.S. and Europe, but immunoreaction and neurotoxicity have been reported. Also, although it has been reported that injection of chymopapain into herniated disc sites gradually restores herniated disc cavities produced by proteolysis, this is attributed to hyperplasia not of the cartilaginous matrix but of fibrous connective tissue. Histological observations of chymopapain-injected canine discs have reportedly revealed replacement of the nucleus pulposus center with fibrocartilaginous tissue (Kudo et al., J. Vet. Med. Sci. 1993, April, 55(2) 211-5). According to experimentation by the present inventors, cases where chymopapain was injected into canine herniated discs showed degradation of the cartilaginous matrix throughout the entire nucleus pulposus and annulus fibrosus, with the surviving chondrocytes markedly reduced and extensively damaged. Thus, lysis of herniated disc with chymopapain is believed to either reduce or eliminate intervertebral disc regenerative capacity. Since chondrocytes maintain their function while suspended in the matrix, the matrix is indispensable for intervertebral disc regeneration. Consideration of these publicly known facts together with knowledge confirmed by the present inventors suggests that in the conventional methods in which proteases are directly administered to herniated disc sites for removal of degenerative nucleus pulposus, it is difficult to sustain the matrix which is required to support the intervertebral disc regenerative capacity of chondrocytes.